Structure-function studies have shown that at least 2 of the 3 regulatory, phosphorylatable serines can be proteolytically removed from the non-helical tailpiece of the Acanthamoeba myosin II heavy chain with essentially no effect on filament formation or phosphorylation-regulatable actin-activated ATPase activity. The positions of the ATP binding site, phosphorylation site and two different actin-binding sites have been localized in Acanthamoeba myosin IA and IB. The phosphorylated amino acid in IA was shown to be threonine while it is serine in IB. The sequence around the PThr has been determined. A third Acanthamoeba myosin I isoform has been purified and characterized. Myosin 1 has been shown to be reversibly bound (Kd approximately 0.05 muM) to the plasma membrane of Acanthamoeba through saturable sites that appear to be independent of actin and from which the myosin can be dissociated at high ionic strength. Studies with Dictyostelium suggest an important role for myosin I at the leading edge of motile cells, a region that is devoid of myosin II but is enriched with actin filaments.